EP2107363B1 - Procédé d'imagerie microscopique à fluorescence d'une structure dans un échantillon avec une forte résolution spatiale tridimensionnelle - Google Patents

Procédé d'imagerie microscopique à fluorescence d'une structure dans un échantillon avec une forte résolution spatiale tridimensionnelle Download PDF

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EP2107363B1
EP2107363B1 EP08006232A EP08006232A EP2107363B1 EP 2107363 B1 EP2107363 B1 EP 2107363B1 EP 08006232 A EP08006232 A EP 08006232A EP 08006232 A EP08006232 A EP 08006232A EP 2107363 B1 EP2107363 B1 EP 2107363B1
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signal
excitation
transfer
fluorophore
sample
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EP2107363A1 (fr
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Johann Dr. Engelhardt
Marion Dr. Lang
Stefan Dr. Hell
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Deutsches Krebsforschungszentrum DKFZ
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Deutsches Krebsforschungszentrum DKFZ
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Definitions

  • the invention relates to a method of fluorescence-microscoplcally imaging a structure In a sample with three-dimensional spatial resolution
  • the invention relates to a method of imaging a structure in a sample comprising the steps of labelling the structure with a fluorophore; subjecting the sample in a spatially limited excitation-volume to an optical excitation signal exciting a portion of the fluorophore located In the excitation-volume for fluorescence; detecting fluorescence light emitted by the excited fluorophore located in and about a centre of maximum intensity of the excitation signal; shifting the centre of maximum intensity of the excitation signal with regard to the sample; and repeating the preceding steps of subjecting and detecting for the shifted centre of maximum intensity of the excitation signal.
  • the fluorescence of the fluorophore In the sample is only excited by two photons of the optical excitation signal each providing a half of the necessary excitation energy.
  • the PSF of the excitation of the fluorophore Is depending on the square of the intensity distribution or of the PSF of the optical excitation signal.
  • the square of the PSF of the optical excitation signal is more concentrated towards the centre of the excitation-volume in all directions, particularly including the axial direction In which the optical excitation signal is focussed into the sample. In this way the spatial resolution in axial direction is directly increased by 2-photon excitation of the fluorophore.
  • a further known measure to increase the axial resolution in fluorescence microscopy is confocal detection of the fluorescence light which means a spatial limitation of the detection volume out of which the fluorescence light is actually detected.
  • the effective PSF is the product of the PSF of the optical excitation signal and of the PSF of the detection arrangement. 2-photon excitation may also be combined with confocal detection.
  • the PSF of the optical excitation signal is spatially confined by means of superimposing coherent parts of the optical excitation signal for forming an interference pattern, the fluorophore substantially only being excited at the maxima of the interference pattern.
  • a prominent type of this kind of fluorescence microscopy is 4Pi microscopy in which spherical wave fronts of the excitation signal are superimposed from two opposite directions in a common focal spot for forming an interference pattern. Within the focal spot, however, not only the first order maximum of the interference pattern is found at the centre but second order maxima flank this first order maximum on both sides of the focal plane. To make use of the full potential of 4Pi microscopy, the second order maxima have to be suppressed In some way.
  • One option Is 2-photon excitation as discussed above.
  • Another option is coherent detection of the fluorescence light both providing a further factor of the total PSF.
  • a further approach of suppressing the fluorescence light from the second order maxima in 4Pi microscopy is forcing the fluorophore within the area of the second order maxima to stimulated emission of fluorescence light which is discriminated from the detected fluorescence light from the sample.
  • STED stimulated emission depletion
  • an excitation-volume in which fluorophore molecules have been excited for fluorescence is confined or reduced In dimensions by de-exciting a part of the fluorophore molecules within the excitation-volume, only leaving the fluorophore molecules within a strongly confined centre of the excitation-volume in their fluorescent state so that the registered fluorescence light can be assigned to the fluorophore molecules within a very small effective excitation-volume.
  • STED fluorescence microscopy The basic principle known from STED fluorescence microscopy is also applied In reversible saturable optical fluorescence transition (RESOLFT) microscopy In which photochromic fluorophores are used to confine the effective excitation-volume in which an optical excitation signal excites a fluorophore for fluorescence. To this end, a switching signal by which the photochromic fluorophore can be switched in an off or dark state and which has a zero point at the centre of the excitation-volume is also applied to the sample.
  • RESOLFT reversible saturable optical fluorescence transition
  • the zero point is a zero point of an Interference pattern, and if the intensity of the switching signal is sufficiently high, the dimensions of the effective excitation-volume in which the fluorophore Is still in its on or fluorescent state and from which the detected fluorescence light can exclusively origin may be kept far below the diffraction barrier (see, for example, US-Patent 7,064.824 ).
  • switchable photochromic fluorophores is also used in photo-activated localization microscopy (PALM) (see WO 2006/127692 A2 ) and stochastic optical reconstruction microscopy (STORM).
  • PAM photo-activated localization microscopy
  • STORM stochastic optical reconstruction microscopy
  • a small proportion of a total concentration of a photochromic fluorophore is switched on into its fluorescent state that the fluorescence light from the single fluorescent fluorophore molecules can be registered separately, and Gaussians are fitted to the PSF of the spatial distribution of the registered fluorescence light to determine the actual location of the fluorescent molecule with sub diffraction spatial resolution.
  • PALM and STORM A strong disadvantage of PALM and STORM is the time consumed by the procedure which limits the observation of living samples, I.e. of objects in which the structure of Interest changes with time, and the production of images which may be directly viewed with the human eye.
  • 2-Photon excitation of fluorophores leads to a 3-dimensional resolution without additional means but the fluorescence yield is comparably low. Additionally a large difference in the wavelengths of the excitation light and the fluorescence light has to be coped with in 2-photon fluorescence microscopy. In confocal laser scanners chromatic aberrations may also be limiting for the precision of the object registration. Here often some axial resolution has to be sacrificed to achieve a large enough signal strength by opening the detection pinhole which additionally requires precise optical alignment.
  • WO 2006/127692 A2 also proposes a way of contributing a third component to the overall point spread function (PSF) by confining the activation illumination for activating photo transformable optical labels (PTOLs) to a localized region in a manner similar to that used to confine the excitation energy itself.
  • PSF point spread function
  • a focussed activation beam is temporally applied to a focal point of a confocal microscope, followed by a focussed beam at the excitation wave lengths for the activated PTOLs, with the resulting emission being detected confocally in a spatially localized manner. This process is then repeated over many voxels to create a complete superresolution 3D image.
  • Dronpa is said to be a particularly good candidate for this method of superresolution, because the activated molecules are returned to the deactivated state by the process of their excitation, thereby providing a natural means to depopulate the activated ensemble while simultaneously determining when the scan should proceed to the next voxel.
  • Ando et al. disclose a method of imaging a structure in a sample labelled with the photochromic fluorophore Dronpa. Via a common objective the sample is subjected to irradiation in a common focal spot both at 405 nm and at 488 nm, and fluorescence light emitted by the fluorophore is confocally detected.
  • the irradiation at 405 nm transfers Dronpa out of a dark or non-fluorescent state into a fluorescence state.
  • the irradiation at 488 nm excites Dronpa for emission of fluorescence light, and returns it into its dark or non-fluorescence state.
  • the new method comprises the steps of selecting a fluorophore from a group of photochromic fluorophores which are repeatedly temporarily transferable by means of an optical transfer signal out of a first into a second photochromic state having specific fluorescence properties, and which display a spontaneous and/or inducible return rate out of the second photochromic state into the first photochromic state; labelling the structure with the fluorophore; via a common objective, subjecting the sample both to the optical transfer signal In a spatially limited transfer-volume, and to an optical excitation signal exciting a portion of the fluorophore being In its second photochromic state for fluorescence in a spatially limited excitation-volume, the transfer-volume and the excitation-volume having a common centre of maximum intensity of the transfer signal and of the excitation signal, wherein, at least in the direction of the optical axis of the common objective, a decrease of Intensity of the transfer signal with the distance to the common centre of maximum Intensity is substantially stronger than any decrease of the effective return rate of
  • the intensity of the transfer signal does not only decrease stronger with the distance to the common centre of maximum Intensity than the effective return rate of the fluorophore in the direction of the optical axis of the common objective but also laterally thereto, i.e. within the focal plane of the common objective.
  • the photochromic fluorophore is transferred into its second photochromic state or switched-on In a transfer-volume having a centre of maximum intensity of the transfer signal coinciding with the centre of maximum intensity of the excitation signal. i. e. the transfer signal and the excitation signal are spatially confined or localized to a common centre of maximum intensity.
  • the detected fluorescence light is only emitted by the fluorophore which is both In its second photochromic state, i.e.
  • the PSF of the emitted fluorescence light Is both depending on the PSF of the transfer signal and the PSF of the excitation signal, i.e. a product of these two PSFs.
  • an Increase of the three-dimensional spatial resolution is obtained in addition to any other measures taken for increasing the spatial resolution
  • a photochromic fluorophore is used as the fluorophore for labelling the structure of interest.
  • the new method may be combined with any further measures for increasing the spatial resolution.
  • the measure of increasing the spatial resolution of the method of the present invention does not decrease the yield of fluorescence light from the sample as known from 2-photon-excitation; and it can be executed without additional equipment as It will be seen from the following description, With suitably selected photochromic fluorophores, it is sufficient to use these fluorophores for labelling the structure of interest in the sample.
  • the group of photochromic fluorophores which may be used in the new method includes photochromic fluorophores which are at least fluorescent in their second photochromic state. They may also be fluorescent in their first photochromic state. In this case, however, there must be some way to discriminate or distinguish the fluorescent light emitted by the fluorophore In its first photochromic state from the fluorescence light emitted by the fluorophore In its second photochromic state. Such a discrimination may, for example, be effected by wavelength selection of the detected fluorescence light (with different emission spectra) or wavelength selection of the optical excitation signal (with different absorption spectra) or appropriate timing of the fluorescence light detection depending on the excitation (with different lifetimes of the excited fluorescent state).
  • the photochromic fluorophores which may be used in the new method are transferred between their first and second photochromic states by cis-/trans-isomerisation, proton transfer, atom group transfer, electron transfer, ring-opening/closing, dimerisation or such, Any Inducible change which changes the spectral properties may be used.
  • photochromic fluorophores which fulfil these requirements.
  • One group of such photochromic fluorophores Is made up by photochromic proteins including mutants of the green fluorescent protein (GFP) like for example DRONPA, Fastlime, switchable Rhodamine derivates etc.
  • Photochromic proteins are particularly suitable as photochromic fluorophores in the new method as they may be expressed by biological samples as a part of the structure of interest in the sample. In this way, no later "staining" of the already existing structure of Interest in the sample is necessary.
  • the fluorophore may be selected from a group of fluorophores which display a substantial spontaneous return rate at ambient temperature of the sample, Even If this condition is met, the lifetime of the fluorophore in its second photochromic state will be comparatively long so that its effective excitation cross-section for excitation by the excitation signal is comparatively large.
  • the lifetime of the second photochromic state of the fluorophore does in no way limit the measuring time in the new method, as the transfer signal may continuously be applied to the sample so that any return rate of the fluorophore out of its second photochromic state into its first photochromic state is compensated for.
  • the ambient temperature of the sample is that temperature at which the relevant steps of the new method are executed. It may be a reduced or even an increased temperature with regard to room temperature.
  • the fluorophore may alternatively or additionally be selected from a from a group of fluorophores which display a substantial return rate inducible by means of a return signal.
  • the return signal may be used after detection of the fluorescence light to completely reset the fluorophore prior to measure the sample at another point of Interest defined by the common centre of maximum Intensity of the transfer signal and the excitation signal. It may, however, also be used simultaneously with the transfer signal to adjust a suitable proportion of the fluorophore consisting of a varying subset of the fluorophore molecules being In Its second state at any relevant point In time.
  • the return signal may be an electric, a magnetic, or any other physical signal. Preferably, however, It is an optical signal like the transfer signal and the excitation signal.
  • the return signal does not have to be a localized signal, and it should not be as localized as the transfer signal as this would be counterproductive in view of the desired distribution of the effective return rate.
  • the sample should be subjected to the return signal in a larger area than the transfer-volume or even everywhere.
  • the sample Is subjected to the optical return signal via the common objective used for the transfer signal and the excitation signal, in contrast to RESOLFT-techniques, however, the intensity of the return signal will have no zero point, preferably no minimum, but most preferably even a maximum at the common centre of maximum intensity of the transfer signal and the excitation signal.
  • a maximum of the return signal at the common centre of maximum intensity Is for example achieved, if the sample is subjected to a uniform intensity distribution of the return signal about the common centre of maximum intensity, as there will be no higher intensity of the return signal than in the common centre of maximum intensity.
  • the excitation-volume may be structured or spatially limited, for example, In that It is defined by a local constructive Interference of a plurality of different parts of the excitation signal. Typically, two parts of the excitation signal are superimposed.
  • the excitation-volume may, however, also be defined by simply focussing the excitation signal from one side into the sample, i.e. by the dimensions of a simple focal spot, or structured Into any single or multi spot, single or multi line, or any other lateral or spatial pattern by any means, Including projection means.
  • the transfer-volume may also be defined by a local constructive interference of a plurality of different parts of the transfer signal.
  • the excitation signal and the transfer signal may generally be directed onto the sample in the same way.
  • the excitation signal and the transfer signal may be both split up in two parts which are superimposed in the transfer-volume and the excitation-volume out of opposite directions through two lenses arranged on opposite sides of the sample,
  • the beam paths of the different parts of both the excitation signal and the transfer signal should differ by less than the coherence length of the excitation signal and the transfer signal to enable the formation of the desired interference pattern with the constructlve interference of both the excitation signal and the transfer signal In the common centre of the excitation-volume and the transfer-volume located about the coinciding focal points of the two lenses.
  • This arrangement corresponds to 4PI-fluorescence microscopy if the aperture angles of the two lenses are sufficiently large.
  • the new method may not only be combined with the known 4PI-fiuoreacence microscopy.
  • a combination with other methods of fluorescence microscopy, in which both a PSF of the excitation of the fluorophore and the PSF of the registration of the fluorescent light from the fluorophore are relevant, is also possible.
  • the excitation signal may excite the fluorophore via a multi-photon process for fluorescence, like for example in 2-photon excitation, and/or the optical transfer signal may transfer the fluorophore into its second photochromic state via a multi-photon process.
  • the new method is combined with confocal detection of the fluorescence light emitted by the fluorophore both being in its second photochromic state and excited by the excitation signal.
  • the fluorescence light may be detected in a spatially limited detection-area having a centre which is confocally arranged with regard to the common centre of maximum intensity of the transfer signal and the excitation signal.
  • the high three-dimensional spatial resolution of the new method due to the multiplication of the PSF of the excitation signal by the PSF of the transfer signal may not only be used in three-dimensionally scanning a sample, although this application of the new method is a preferred embodiment.
  • the high three-dlmensional spatial resolution of the method of the present invention is a substantial advantage, even If the sample is only scanned in one or two dimensions, For three-dimensionally scanning the sample, the common centre of maximum intensity of the transfer signal and the excitation signal is shifted with regard to the sample in three linearly independent directions to completely cover at least one area of the sample extending In all three dimensions.
  • both the transfer signal and the excitation signal are limited to a spatially limited volume.
  • the benefits resitting from the effective PSF of the excitation of the fluorophore In the new method being a product of the PSF of the transfer signal and the PSF of the excitation signal, however, are only maximum In a spatial direction of interest, If the transfer signal and the excitation signal have essentially a same width at half maximum in that direction about the common centre of maximum intensity of the transfer signal and the excitation signal. Otherwise, the product of both individual PSFs is not significantly localized over the already more localized individual PSF.
  • the new method is not depending on any of the optical signals used being pulsed signals. Instead, all optical signals to which the sample is subjected may be continuous wave signals. However, the use of pulsed optical signals Is also possible.
  • the return signal may be a pulsed signal completely returning the fluorophore into the first photochromic state prior to the steps of subjecting the sample to the transfer signal and the excitation signal and of detecting fluorescence light.
  • This strong return signal strongly contributes to the relevant time-averaged spatial distribution of the effective return rate of the fluorophore, in fact, it ensures that the effective return rate of the fluorophore decreases less with the distance to the common centre of maximum intensity of the transfer signal and the excitation signal than the intensity of the transfer signal as required in the new method, even if the excitation signal, for example, induces a localized contribution to the effective return rate which as strongly decreases with the distance to the common centre of maximum intensity as the Intensity of the transfer signal.
  • the absorption spectra of known photochromic fluorophores suitable for use in the present invention often display an absorption peak for the excitation signal that shows overlapping with an absorption peak for the transfer signal or for a return signal.
  • the transfer signal and the excitation signal or the return signal and the excitation signal may be identical, If they have a wavelength within one of these overlaps.
  • the execution of the new method is particularly simple In that it only requires the selection of an appropriate photochromic fluorophore to achieve the additionally three-dimensionally limited effective PSF of the excitation of the fluorophore for fluorescence,
  • a standard fluorescence microscope particularly a fluorescence microscope with confocal detection of the fluorescence light emitted from a sample may be easily upgraded for threa-dimensionally imaging a sample in that a mechanism is provided which allows for additionally axially shifting the focal spot of one beam of light both providing the transfer signal and the excitation signal within the sample.
  • the fluorescence microscope 1 depicted in Fig. 1 is used for three-dimenslonally imaging a structure in a sample 2.
  • the sample 2 is arranged on a sample carrier 3 suitable for displacing the sample 2 In the axial direction of an objective 4.
  • the objective 4 Is used for focussing both an optical excitation signal 5 coming from an excitation laser 6 and an optical transfer signal 7 coming from a transfer laser 8 Into a common focal spot within the sample 2, As the excitation signal 5 and the transfer signal 7 may have the same wavelength
  • the additional transfer laser 8 is optional.
  • a return laser 9 supplying an optional return signal 10 which is focussed in the pupil of the objective 4 may be provided.
  • Dichroitic mirrors 11 are used to combine any optical signals 5, 7, and 10 onto one common optical axis.
  • a xy-scanner laterally shifts the common optical axis of the optical signals 5, 7, and 10 to scan the sample 2 with the common focal spot of these optical signals within the focal plane of the objective 4,
  • the third dimension for three-dlmensionelly scanning the sample 2 is provided by the sample carrier 3.
  • a fluorophore in the sample 2 is excited for fluorescence.
  • the fluorescence light 18 spontaneously emitted by the fluorophore In the sample 2 is detected via the objective 4 by a CCD-camera 13, a dichroitic mirror 14 reflecting the fluorescence light away from the optical signals 5 and 7.
  • the CCD-camera 13 may be used to simulate a confocal detection of the fluorescence light 18 emitted out of the focal spot in the sample 2, or a confocal pinhole (not depicted here) may be provided to this end.
  • the fluorophore may not only be stimulated for fluorescence in one common focal spot or line having a common centre of maximum intensity of the optical signals 5 and 7 but in a plurality of such common spots or lines sufficiently separated for separately registering the fluorescence light emitted out of these spots with the CCD-camera 13.
  • the block diagram of Fig. 2 refera to the basic functional units required for executing the new method like, for example, with the fluorescence microscope 1 according to Fig. 1 .
  • a light source 15 provides the optical signals 5, 7, and 10, If required,
  • the light source 15 may include the lasers 6, 8 and 9 according to Fig. 1 .
  • These lasers may be pulsed lasers or continuous wave lasers.
  • the lasers may be single line lasers. Instead of two different lasers a multiline laser may be used, or even only one line of a multiline loser may be selected.
  • a white light laser may also be suitable,
  • the light source 15 may comprise LEDs, bulbs, MP-lasers or any other light sources with appropriate wavelengths and powers of the emitted light, which are required for transferring, exciting and/or returning the fluorophore in the sample 2.
  • the optical signals 5, 7, 10 are supplied by the light source 15 to a structuring/scanning and detection unit 16.
  • This unit 16 may form single or multiple spots or lines or any focusable structure out of the optical signals 5 and 7, while it more uniformly distributes the return signal 10, and shifts the spatial position of all these signals 5, 7, and 10 to scan the sample 2.
  • the unit 18 confocally detects the fluorescence light emitted by the sample 2.
  • the unit 16 includes the xy-scanner 12, the sample carrier 3, the dichroitic mirror 14 and the CCD-camera 13 according to Fig. 1 .
  • a light microscope 17 is provided to project the structure formed by the unit 16 into the sample 2 and to provide the fluorescence light from the sample 2.
  • This light microscope may be a widefield microscope, a 4PI- microscope or any other suitable light microscope.
  • the objective 4 according to Fig, 1 is part of the light microscope 17.
  • the three-dimensional optical resolution in imaging a structure In the sample 2 labelled with a fluorophore essentially is the product of an excitation PSF (point spread function) defining an effective excitation-volume in which the fluorophore is excited for fluorescence multiplied by a detection PSF defining the detection-volume out of which fluorescence light emitted by the fluorophore in the sample 2 is detected.
  • the detection PSF depends on the setup of the light microscope 17 and of the detection part of the unit 16 according to Fig. 2 .
  • the effective excitation PSF also depends on the light microscope 17 and on the structuring part of the unit 16 according to Fig. 2 .
  • the effective excitation PSF depends on the inherent properties of the fluorophore.
  • the fluorophore used for labelling a structure of interest In the sample 2 is a photochromic fluorophore which is transferred out of a first photochromic state into a second photochromic state by means of the optical transfer signal 7 according to Fig. 1 .
  • the optical transfer signal 7 may have the same wavelength as the optical excitation signal 5. Nevertheless, the optical transfer signal 7 has a different function than the optical excitation signal 5. If the photochromic fluorophore in the sample 2 is in its first photochromic state it does not emit any substantial fluorescence light or it does at least not emit that fluorescence light 18 which is registered by the CCD-camera 13 according to Fig. 1 .
  • the effective excitation PSF for the photochromic fluorophore is the product of the PSF of the optical transfer signal 7 within the sample 2 and of the PSF of the optical excitation signal 5 in the sample 2.
  • the spatial resolution in Imaging the sample 2 is increased due to the inherent properties of the photochromic fluorophore in a similar way as In 2-photon excitation of a fluorophore In 2-photon fluorescence microscopy.
  • the new method using the photochromic fluorophore avoids a number of strong drawbacks of 2-photon fluorescence microscopy.
  • the yield of fluorescence light from a certain number of molecules of the photochromic fluorophore is much higher in the method according to the present invention than with 2-photon excitation of a fluorophore.
  • the wavelength of the optical excitation signal 5 and the optical transfer signal 7, which may even be identical, are much closer to the wavelength of the fluorescence light 18 in the new method than with 2-photon excitation.
  • the objective 4 according to Fig. 1 or the light microscope 17 according to Fig. 2 has not to be compensated for chromatic errors due to strongly differing wavelengths.
  • the light sources suitable for 2-photon excitation of fluorophores suitable for fluorescence microscopy are complicated and expensive due to the short pulses at long wavelengths required for 2-photon excitation.
  • the light source 15 according to Fig. 2 or the lasers 6 and 8 according to Fig. 1 may simply work in the usual wavelength range for 1-photon excitation and may thus be quite low cost.
  • Fig. 3 Illustrates a cycle of the states through which a molecule of the photochromic fluorophore 19 passes during the steps of the method of the present invention.
  • the fluorophore 19 is in its first photochromic state 20.
  • the fluorophore In this first photochromic state 20 the fluorophore is not excitable for the emission of that fluorescence light 18 which is of interest here.
  • the fluorophore 19 is transferred into its second photochromic state 21.
  • the fluorophore 19 is excitable for fluorescence.
  • the second photochromic state 21 may be called the fluorescent state of the fluorophore 19.
  • the fluorophore 19 may absorb a photon of the optical excitation signal 5 to get into an excited second photochromic state 22.
  • This excited second photochromic state 22 has a very short life time as compared to the lifetime of the second photochromic state 21, the excited second state 22 being an energetic sub-state of the second photochromic state 21.
  • the fluorophore 19 returns into its second photochromic state 21 upon spontaneous emission of a photon of fluorescence light 18. This fluorescence light 18 is detected in the new method to image a structure of Interest labelled with the fluorophore 19.
  • the fluorophore 19 may be again excited for fluorescence by another photon of the excitation signal 5.
  • the number of fluorescence cycles including the absorption of a photon of the optical excitation signal 5 and the emission of a photon of florescence light 18 during the life time of the second photochromic state 21 of the fluorophore 19 may be quite high.
  • the life time of the second photochromic state 21 of the fluorophore 19 may be limited by thermal effects, i.e. by a thermal excitation of the fluorophore 19 for returning into its first photochromic state 20.
  • FSM Fluorescence Switching Microscopy
  • Fluorescence switching microscopy enables optical sectioning without requiring confocal optics or 2-photon excitation.
  • FSM Fluorescence switching microscopy
  • fluorescence microscopy Since the development of fluorescence microscopy it has played an ever increasing role in biological research. But it was not until the invention of the confocal microscope in 1957 that a 3-dimensional optical sectioning could be obtained. Before that, fluorescence microscopy was limited to discriminating objects in the lateral direction only.
  • Confocal microscopy overcomes this limitation by scanning the object by a point of light and using a pinhole in the detection path which leads to a pointlike excitation and detection point spread function (PSF).
  • PSD pointlike excitation and detection point spread function
  • the PSFs are given by the square of the electromagnetic fields of excitation and detection, respectively, and can be calculated according to a vectorial theory by Richards and Wolf: h eff ⁇
  • p ( r ) denotes the pinhole function, which is 1 for an infinitely small pinhole, i.e. pointlike detection.
  • the product of the two PSFs leads to a spatial discrimination of the fluorescence signal along the optical ( z ) axis.
  • the axial FWHM in a confocal microscope typically amounts to 500 nm.
  • the axial discrimination in confocal microscopy and 2-photon microscopy can be generalized: whenever at least two localized PSFs are multiplied in the imaging process, be it illumination PSF and excitation PSF, or two times the illumination PSF, an axial discrimination and therefore a 3-dimensional resolution can be achieved. If even more PSFs of focusing beams contribute to the effective PSF, the resolution in all directions gets even better, due to the multiplication of the contributing distributions.
  • FSM fluorescence switching microscopy
  • the optical transfer function (OTF) of a microscope is given by the fourier transform of the PSF.
  • OTF features the so-called "missing cone", where no spatial frequencies along the optical axis are transmitted. This prevents the axial discrimination of laterally extended object structures.
  • the missing cone is not present, if the PSF describing the imaging properties contains the product of at least two focused beams of light.
  • the illumination structure may be a single or multiple spots or lines or other preferably focussed structures.
  • a scanned spot with switching at 405nm, and excitation at 488nm and a fluorescence at around 515nm theoretically features an axial FWHM of 231 nm using a 1.46NA Oil immersion objective. Note these results are valid in general as long as switching and excitation take place simultaneously with preferably diffraction limited illumination structures.
  • the selected molecule can be reversibly switched between a fluorescent or active or on state and a dark or inactive or off state.
  • optical sectioning is achieved only in those cases in which the concentration of active molecules is decreasing with the distance from the focal region.
  • n on z b ⁇ P act z 2 + k spon c ⁇ P exc z 2 + k spoff
  • switch-able fluorescent proteins such as DRONPA and its mutants and switchable organic dyes such as SpaRhodamines and others became available recently (Stiel et al, Ando et al., Fölling et al.). Ando et al. show cells labeled with switch-able dyes which display fluorescence only, if both switching and excitation light are applied simultaneously. Ando et al. used a dye which quickly returned into its active state. So they had to use a confocal microscope to suppress the out of focus light. Ando et al. actually suggest the use of switch-able dyes for wide field detection when two different objectives are used for the two lasers. In our case both lasers can use the same beam paths, which leads to a much simpler optical setup.
  • Still another class of dyes such as the ASCP type are switched on at the excitation wavelength and are switched off in the near UV range.
  • rate equations must be modified accordingly, here.
  • Dr31 can be activated in the UV-range (at around 400nm), or alternatively with two-photon activation at around 800 nm.
  • the excitation takes place at 488 nm, and the fluorescence is 515nm.
  • the readout process at 488 nm simultaneously switches the DRONPA-mutant into its off-state.
  • Fig. 4 shows intensity profiles along the optical axis of the thin layer of fluorescence dyes detected by a non confocal large area detector.
  • the intensity profiles indicate that the switching of Dronpa delivers depth discrimination even In laterally extended structures which is not present in the control experiment using regular GFP fluorescent dyes. Note that the slow drop the intensity in the GFP case is due to the finite beam diameters in the microscope setup.
  • the presented data is just an example of the new general method enabled by the recent advent of photo-stable, fast-switching fluorescent markers.
  • fluorescent proteins offers the possibility for fast 3D in-vivo Imaging, especially when the sample is illuminated by multiple spots or lines or other preferably diffraction limited structures. These may be generated by a variety of methods using devices such as micro lens arrays, SLMs, LED or DMD projectors, LED arrays, holographic pattern generators etc.
  • activation can also be accomplished by 2-photon absorption at 800nm simultaneously with a 488nm excitation or other wavelengths from new dyes which may become avaliable.
  • the method of using photo switch-able dye molecules offers a new quality in high resolution 3d imaging In 4PI with respect to probe size, side-lobe reduction and imaging speed. 4PI microscopy is boosted by the additional kick of switching which reduces the effective detection volume to less than 1 atto liter which will be especially helpful in 3-dimensionally dons structures.
  • FSM may be advantageously used in many other microscopy techniques such as high throughput applications where it also suppresses effectively light generated not in the focus.

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Claims (15)

  1. Procédé d'imagerie d'une structure dans un échantillon (2) avec une résolution spatiale tridimensionnelle, le procédé comprenant les étapes ci-dessous consistant à :
    - sélectionner un fluorophore (19) à partir d'un groupe de fluorophores photochromiques qui peuvent être transférés, de manière répétée et temporaire, au moyen d'un signal de transfert optique (7), d'un premier état photochromique (20) vers un second état photochromique (21), ayant des propriétés de fluorescence spécifiques au second état photochromique (21), et qui affichent un taux de retour du second état photochromique (21) au premier état photochromique (20), le taux de retour étant spontané et/ou pouvant être induit au moyen d'un signal de retour physique (10) ;
    - étiqueter la structure avec le fluorophore (19) ;
    - par l'intermédiaire d'un objectif commun (4), focaliser le signal de transfert optique (7) en vue de soumettre l'échantillon (2), dans un volume de transfert limité spatialement, au signal de transfert optique (7) qui transfère une partie du fluorophore (19) située dans le volume de transfert vers son second état photochromique (21) ;
    - par l'intermédiaire de l'objectif commun (4), focaliser un signal d'excitation optique (5) en vue de soumettre l'échantillon (2), dans un volume d'excitation limité spatialement, au signal d'excitation optique (5) qui excite une partie du fluorophore (19) située dans le volume d'excitation, et étant dans son second état photochromique (21) de fluorescence, le volume de transfert et le volume d'excitation présentant un centre commun d'intensité maximale du signal de transfert (7) et du signal d'excitation (5) ;
    - dans lequel, un taux de retour efficace du fluorophore (19), du second état photochromique (21) au premier état photochromique (20), est dominé par le taux de retour spontané, ou est modifié par le signal de retour physique (10), en vue de n'afficher qu'une dépendance faible quant à la distance jusqu'au centre commun d'intensité maximale, de sorte que, au moins dans la direction de l'axe optique de l'objectif commun (4), une diminution de l'intensité du signal de transfert focalisé avec la distance jusqu'au centre commun d'intensité maximale est sensiblement plus forte qu'une quelconque diminution du taux de retour efficace du fluorophore (19) avec la distance jusqu'au centre commun d'intensité maximale ;
    - détecter une lumière fluorescente (18), émise par le fluorophore excité (19) dans son second état photochromique (21), située au sein et autour du centre commun d'intensité maximale du signal de transfert (7) et du signal d'excitation (5) ;
    - décaler le centre commun d'intensité maximale du signal de transfert (7) et du signal d'excitation (5) relativement à l'échantillon (2) ; et
    - répéter les étapes précédentes de soumission et de détection pour le centre commun d'intensité maximale décalé du signal de transfert (7) et du signal d'excitation (5).
  2. Procédé selon la revendication 1, caractérisé en ce que le groupe de fluorophores photochromiques comprend des fluorophores photochromiques qui sont au moins fluorescents dans leur second état photochromique, et qui sont transférés entre leurs premiers états photochromiques (20) et leurs seconds états photochromiques (21) par le biais de l'un des procédés ci-dessous :
    - cis/trans-isomérisation ;
    - transfert de protons ;
    - transfert de groupes d'atomes ;
    - transfert d'électrons ;
    - ouverture / fermeture de cycles ; et
    - dimérisation.
  3. Procédé selon la revendication 1 ou 2, caractérisé en ce que le fluorophore (19) est sélectionné parmi un groupe de fluorophores qui présentent un taux de retour spontané considérable à la température ambiante de l'échantillon (2).
  4. Procédé selon la revendication 1, 2 ou 3, caractérisé en ce que le fluorophore (19) est sélectionné parmi un groupe de fluorophores qui présentent un taux de retour inductible considérable au moyen d'un signal de retour optique (10).
  5. Procédé selon la revendication 4, caractérisé en ce que l'échantillon est soumis au signal de retour (10) par l'intermédiaire de l'objectif commun, dans lequel l'intensité du signal de retour (10) ne présente pas de valeur ponctuelle nulle, ne présente de préférence pas de valeur minimale, et présente de préférence une valeur maximale au niveau du centre commun d'intensité maximale.
  6. Procédé selon la revendication 4 ou 5, caractérisé en ce que l'échantillon est soumis à une distribution d'intensité uniforme du signal de retour (10) autour du centre commun d'intensité maximale.
  7. Procédé selon l'une quelconque des revendications 1 à 6, caractérisé en ce que le volume d'excitation est défini par une interférence constructive locale d'une pluralité de différentes parties du signal d'excitation (5).
  8. Procédé selon l'une quelconque des revendications 1 à 7, caractérisé en ce que le volume de transfert est défini par une interférence constructive locale d'une pluralité de différentes parties du signal de transfert (7).
  9. Procédé selon les revendications 7 et 8, caractérisé en ce que le signal d'excitation (5) et le signal de transfert (7) sont tous deux divisés en deux parties qui sont superposées dans le volume de transfert et le volume d'excitation à partir de directions opposées à travers deux objectifs communs agencés sur des côtés opposés de l'échantillon, les chemins de faisceau des différentes parties du signal d'excitation et du signal de transfert différant mutuellement, en termes de longueurs, d'une quantité inférieure à celles des longueurs de cohérence du signal d'excitation et du signal de transfert.
  10. Procédé selon l'une quelconque des revendications 1 à 9, caractérisé en ce que la lumière fluorescente (18) est détectée dans une zone de détection limitée spatialement présentant un centre qui est agencé de manière homofocale relativement au centre commun d'intensité maximale du signal de transfert (7) et du signal d'excitation (5).
  11. Procédé selon l'une quelconque des revendications 1 à 10, caractérisé en ce que l'échantillon (2) est balayé de manière tridimensionnelle avec le centre commun d'intensité maximale du signal de transfert (7) et du signal d'excitation (5).
  12. Procédé selon l'une quelconque des revendications 1 à 11, caractérisé en ce que le signal de transfert (7) et le signal d'excitation (5) présentent une même largeur à mi-hauteur au niveau du centre commun d'intensité maximale du signal de transfert (7) et du signal d'excitation (5).
  13. Procédé selon l'une quelconque des revendications 1 à 12, caractérisé en ce que tous les signaux optiques (5, 7, 10) auxquels l'échantillon est soumis sont des signaux à ondes entretenues.
  14. Procédé selon l'une quelconque des revendications 1 à 12, caractérisé en ce que le signal de retour (10) est un signal par impulsions renvoyant totalement le fluorophore (19) au premier état photochromique (20) préalablement aux étapes consistant à soumettre l'échantillon au signal de transfert et au signal d'excitation, et à détecter une lumière fluorescente.
  15. Procédé selon l'une quelconque des revendications 1 à 14, caractérisé en ce que le signal de transfert (7) et le signal d'excitation (5) sont identiques.
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